Strategies used by nurses regarding underreporting of rural work accidents due to pesticide use / Estrategias utilizadas por los enfermeros en relación con el subregistro de accidentes laborales rurales causados por el uso de plaguicidas / Estratégias utilizadas por enfermeiros sobre subnotificações de acidentes de trabalho rural por uso de agrotóxicos

ABSTRACT Objective: To learn the strategies used regarding underreporting of pesticide use in rural areas. Methods: A qualitative study was carried out in eight primary healthcare units in rural areas and two emergency care units in a municipality in southern Brazil. Data collection took place in 2023 through interviews. Twenty professional nurses participated. The data was submitted to content analysis. Results: The strategies identified were lifelong and continuing education for the professionals who carry out the notification, active search and training of workers who deal directly with this type of substance, computerizing the notification by filling in the forms online, and carrying out research on the subject. Final considerations: Nurses play an important role in reporting occupational accidents caused by the use of pesticides, improving occupational safety in rural areas.

RESUMEN Objetivo: Conocer las estrategias utilizadas para el subregistro del uso de plaguicidas en áreas rurales. Métodos: Estudio cualitativo realizado en ocho unidades de atención primaria en áreas rurales y dos unidades de atención de emergencia en un municipio del sur de Brasil. La recopilación de datos se realizó en 2023 mediante entrevistas. Participaron 20 profesionales de enfermería. Los datos fueron sometidos a análisis de contenido. Resultados: Las estrategias identificadas fueron la educación continua y permanente de los profesionales que realizan la notificación, la búsqueda activa y la capacitación de los trabajadores que tratan directamente con este tipo de sustancia, la informatización de la notificación mediante el llenado de los formularios en línea y la realización de investigaciones sobre el tema. Consideraciones finales: Los enfermeros desempeñan un papel importante en la notificación de accidentes laborales causados por el uso de plaguicidas, mejorando la seguridad laboral en las zonas rurales.

RESUMO Objetivo: Conhecer as estratégias utilizadas acerca das subnotificações por uso de agrotóxicos no meio rural. Método: Estudo qualitativo realizado em oito unidades básicas de regiões rurais e duas unidades de pronto atendimento de um município do sul do Brasil. A coleta de dados deu-se em 2023, por entrevistas. Participaram 20 profissionais enfermeiros. Os dados foram submetidos à análise de conteúdo. Resultados: Apontaram como estratégias a realização da educação permanente e continuada dos profissionais que realizam a notificação, a busca ativa e a capacitação dos trabalhadores que lidam diretamente com esse tipo de substância, a informatização da notificação por meio do preenchimento das fichas online e a realização de pesquisas acerca da temática. Considerações Finais: Os enfermeiros desempenham um papel importante na notificação de acidentes de trabalho pelo uso de agrotóxicos, melhorando a segurança ocupacional no meio rural.

Mitochondria Transplantation Mitigates Damage in an In Vitro Model of Renal Tubular Injury and in an Ex Vivo Model of DCD Renal Transplantation.

OBJECTIVES: To test whether mitochondrial transplantation (MITO) mitigates damage in 2 models of acute kidney injury (AKI). BACKGROUND: MITO is a process where exogenous isolated mitochondria are taken up by cells. As virtually any morbid clinical condition is characterized by mitochondrial distress, MITO may find a role as a treatment modality in numerous clinical scenarios including AKI. METHODS: For the in vitro experiments, human proximal tubular cells were damaged and then treated with mitochondria or placebo. For the ex vivo experiments, we developed a non-survival ex vivo porcine model mimicking the donation after cardiac death renal transplantation scenario. One kidney was treated with mitochondria, although the mate organ received placebo, before being perfused at room temperature for 24 hours. Perfusate samples were collected at different time points and analyzed with Raman spectroscopy. Biopsies taken at baseline and 24 hours were analyzed with standard pathology, immunohistochemistry, and RNA sequencing analysis. RESULTS: In vitro, cells treated with MITO showed higher proliferative capacity and adenosine 5'-triphosphate production, preservation of physiological polarization of the organelles and lower toxicity and reactive oxygen species production. Ex vivo, kidneys treated with MITO shed fewer molecular species, indicating stability. In these kidneys, pathology showed less damage whereas RNAseq analysis showed modulation of genes and pathways most consistent with mitochondrial biogenesis and energy metabolism and downregulation of genes involved in neutrophil recruitment, including IL1A, CXCL8, and PIK3R1. CONCLUSIONS: MITO mitigates AKI both in vitro and ex vivo.

Identification and Characterization of the Wilms Tumor Cancer Stem Cell.

A nephrogenic progenitor cell (NP) with cancer stem cell characteristics driving Wilms tumor (WT) using spatial transcriptomics, bulk and single cell RNA sequencing, and complementary in vitro and transplantation experiments is identified and characterized. NP from WT samples with NP from the developing human kidney is compared. Cells expressing SIX2 and CITED1 fulfill cancer stem cell criteria by reliably recapitulating WT in transplantation studies. It is shown that self-renewal versus differentiation in SIX2+CITED1+ cells is regulated by the interplay between integrins ITGß1 and ITGß4. The spatial transcriptomic analysis defines gene expression maps of SIX2+CITED1+ cells in WT samples and identifies the interactive gene networks involved in WT development. These studies define SIX2+CITED1+ cells as the nephrogenic-like cancer stem cells of WT and points to the renal developmental transcriptome changes as a possible driver in regulating WT formation and progression.

The spatially resolved transcriptional profile of acute T cell-mediated rejection in a kidney allograft.

Analysis of the transcriptional profile of graft biopsies represents a promising strategy to study T cell-mediated-rejection (TCMR), also known as acute cellular rejection. However, bulk RNA sequencing of graft biopsies may not capture the focal nature of acute rejection. Herein, we used the whole exome GeoMX Digital Space Profiling platform to study five tubular and three glomerular regions of interest in the kidney graft biopsy from a patient with a chronic-active TCMR episode and in analogous areas from two different normal kidney control biopsies. All kidney sections were from paraffin blocks. Overall, inflammatory genes were significantly upregulated in the tubular areas of the TCMR biopsy and showed an enrichment for gene-ontology terms associated with T-cell activation, differentiation, and proliferation. Enrichment analysis of the 100 genes with the highest coefficient of variation across the TCMR tubular regions of interest revealed that these highly variable genes are involved in kidney development and injury and interestingly do not associate with the 2019 Banff classification pathology scores within the individual regions of interest. Spatial transcriptomics allowed us to unravel a previously unappreciated variability across different areas of the TCMR biopsy related to the graft response to the alloimmune attack, rather than to the immune cells. Thus, our approach has the potential to decipher clinically relevant, new pathogenic mechanisms, and therapeutic targets in acute cellular rejection and other kidney diseases with a focal nature.

CyTOF-Enabled Analysis Identifies Class-Switched B Cells as the Main Lymphocyte Subset Associated With Disease Relapse in Children With Idiopathic Nephrotic Syndrome.

B cell depleting therapies permit immunosuppressive drug withdrawal and maintain remission in patients with frequently relapsing nephrotic syndrome (FRNS) or steroid-dependent nephrotic syndrome (SDNS), but lack of biomarkers for treatment failure. Post-depletion immune cell reconstitution may identify relapsing patients, but previous characterizations suffered from methodological limitations of flow cytometry. Time-of-flight mass cytometry (CyTOF) is a comprehensive analytic modality that simultaneously quantifies over 40 cellular markers. Herein, we report CyTOF-enabled immune cell comparisons over a 12-month period from 30 children with SDNS receiving B cell depleting therapy who either relapsed (n = 17) or remained stable (n = 13). Anti-CD20 treatment depleted all B cells subsets and CD20 depleting agent choice (rituximab vs ofatumumab) did not affect B cell subset recovery. Despite equal total numbers of B cells, 5 subsets of B cells were significantly higher in relapsing individuals; all identified subsets of B cells were class-switched. T cell subsets (including T follicular helper cells and regulatory T cells) and other major immune compartments were largely unaffected by B cell depletion, and similar between relapsing and stable children. In conclusion, CyTOF analysis of immune cells from anti-CD20 antibody treated patients identifies class-switched B cells as the main subset whose expansion associates with disease relapse. Our findings set the basis for future studies exploring how identified subsets can be used to monitor treatment response and improve our understanding of the pathogenesis of the disease.

Placental extracellular vesicles-associated microRNA-519c mediates endotoxin adaptation in pregnancy.

BACKGROUND: Pregnancy represents a unique challenge for the maternal-fetal immune interface, requiring a balance between immunosuppression, which is essential for the maintenance of a semiallogeneic fetus, and proinflammatory host defense to protect the maternal-fetal interface from invading organisms. Adaptation to repeated inflammatory stimuli (endotoxin tolerance) may be critical in preventing inflammation-induced preterm birth caused by exaggerated maternal inflammatory responses to mild or moderate infections that are common during pregnancy. However, the exact mechanisms contributing to the maintenance of tolerance to repeated infections are not completely understood. MicroRNAs play important roles in pregnancy with several microRNAs implicated in gestational tissue function and in pathologic pregnancy conditions. MicroRNA-519c, a member of the chromosome 19 microRNA cluster, is a human-specific microRNA mainly expressed in the placenta. However, its role in pregnancy is largely unknown. OBJECTIVE: This study aimed to explore the role of "endotoxin tolerance" failure in the pathogenesis of an exaggerated inflammatory response often seen in inflammation-mediated preterm birth. In this study, we investigated the role of microRNA-519c, a placenta-specific microRNA, as a key regulator of endotoxin tolerance at the maternal-fetal interface. STUDY DESIGN: Using a placental explant culture system, samples from term and second-trimester placentas were treated with lipopolysaccharide. After 24 hours, the conditioned media were collected for analysis, and the placental explants were re-exposed to repeated doses of lipopolysaccharide for 3 days. The supernatant was analyzed for inflammatory markers, the presence of extracellular vesicles, and microRNAs. To study the possible mechanism of action of the microRNAs, we evaluated the phosphodiesterase 3B pathway involved in tumor necrosis factor alpha production using a microRNA mimic and phosphodiesterase 3B small interfering RNA transfection. Finally, we analyzed human placental samples from different gestational ages and from women affected by inflammation-associated pregnancies. RESULTS: Our data showed that repeated exposure of the human placenta to endotoxin challenges induced a tolerant phenotype characterized by decreased tumor necrosis factor alpha and up-regulated interleukin-10 levels. This reaction was mediated by the placenta-specific microRNA-519c packaged within placental extracellular vesicles. Lipopolysaccharide treatment increased the extracellular vesicles that were positive for the exosome tetraspanin markers, namely CD9, CD63, and CD81, and secreted primarily by trophoblasts. Primary human trophoblast cells transfected with a microRNA-519c mimic decreased phosphodiesterase 3B, whereas a lack of phosphodiesterase 3B, achieved by small interfering RNA transfection, led to decreased tumor necrosis factor alpha production. These data support the hypothesis that the anti-inflammatory action of microRNA-519c was mediated by a down-regulation of the phosphodiesterase 3B pathway, leading to inhibition of tumor necrosis factor alpha production. Furthermore, human placentas from normal and inflammation-associated pregnancies demonstrated that a decreased placental microRNA-519c level was linked to infection-induced inflammatory pathologies during pregnancy. CONCLUSION: We identified microRNA-519c, a human placenta-specific microRNA, as a novel regulator of immune adaptation associated with infection-induced preterm birth at the maternal-fetal interface. Our study serves as a basis for future experiments to explore the potential use of microRNA-519c as a biomarker for infection-induced preterm birth.

Persistent COUP-TFII expression underlies the myopathy and impaired muscle regeneration observed in resistance to thyroid hormone-alpha.

Thyroid hormone signaling plays an essential role in muscle development and function, in the maintenance of muscle mass, and in regeneration after injury, via activation of thyroid nuclear receptor alpha (THRA). A mouse model of resistance to thyroid hormone carrying a frame-shift mutation in the THRA gene (THRA-PV) is associated with accelerated skeletal muscle loss with aging and impaired regeneration after injury. The expression of nuclear orphan receptor chicken ovalbumin upstream promoter-factor II (COUP-TFII, or Nr2f2) persists during myogenic differentiation in THRA-PV myoblasts and skeletal muscle of aged THRA-PV mice and it is known to negatively regulate myogenesis. Here, we report that in murine myoblasts COUP-TFII interacts with THRA and modulates THRA binding to thyroid response elements (TREs). Silencing of COUP-TFII expression restores in vitro myogenic potential of THRA-PV myoblasts and shifts the mRNA expression profile closer to WT myoblasts. Moreover, COUP-TFII silencing reverses the transcriptomic profile of THRA-PV myoblasts and results in reactivation of pathways involved in muscle function and extracellular matrix remodeling/deposition. These findings indicate that the persistent COUP-TFII expression in THRA-PV mice is responsible for the abnormal muscle phenotype. In conclusion, COUP-TFII and THRA cooperate during post-natal myogenesis, and COUP-TFII is critical for the accelerated skeletal muscle loss with aging and impaired muscle regeneration after injury in THRA-PV mice.

A Comprehensive Phenotypic and Functional Immune Analysis Unravels Circulating Anti-Phospholipase A2 Receptor Antibody Secreting Cells in Membranous Nephropathy Patients.

INTRODUCTION: Primary membranous nephropathy (MN) is characterized by the presence of antipodocyte antibodies, but studies describing phenotypic and functional abnormalities in circulating lymphocytes are limited. METHODS: We analyzed 68 different B- and T-cell subsets using flow cytometry in 30 MN patients (before initiating immunosuppression) compared with 31 patients with non-immune-mediated chronic kidney disease (CKD) and 12 healthy individuals. We also measured 19 serum cytokines in MN patients and in healthy controls. Lastly, we quantified the ex vivo production of phospholipase A2 receptor (PLA2R)-specific IgG by plasmablasts (measuring antibodies in culture supernatants and by the newly developed FluoroSpot assay [AutoImmun Diagnostika, Strasberg, Germany]) and assessed the circulating antibody repertoire by phage immunoprecipitation sequencing (PhIP-Seq). RESULTS: After adjusting for multiple testing, plasma cells and regulatory B cells (BREG) were significantly higher (P < 0.05) in MN patients compared with both control groups. The percentages of circulating plasma cells correlated with serum anti-PLA2R antibody levels (P = 0.042) and were associated with disease activity. Ex vivo-expanded PLA2R-specific IgG-producing plasmablasts generated from circulating PLA2R-specific memory B cells (mBCs) correlated with serum anti-PLA2R IgG antibodies (P < 0.001) in MN patients. Tumor necrosis factor-α (TNF-α) was the only significantly increased cytokine in MN patients (P < 0.05), whereas there was no significant difference across study groups in the autoantibody and antiviral antibody repertoire. CONCLUSION: This extensive phenotypic and functional immune characterization shows that autoreactive plasma cells are present in the circulation of MN patients, providing a new therapeutic target and a candidate biomarker of disease activity.

Glomerular endothelial cell heterogeneity in Alport syndrome.

Glomerular endothelial cells (GEC) are a crucial component of the glomerular physiology and their damage contributes to the progression of chronic kidney diseases. How GEC affect the pathology of Alport syndrome (AS) however, is unclear. We characterized GEC from wild type (WT) and col4α5 knockout AS mice, a hereditary disorder characterized by progressive renal failure. We used endothelial-specific Tek-tdTomato reporter mice to isolate GEC by FACS and performed transcriptome analysis on them from WT and AS mice, followed by in vitro functional assays and confocal and intravital imaging studies. Biopsies from patients with chronic kidney disease, including AS were compared with our findings in mice. We identified two subpopulations of GEC (dimtdT and brighttdT) based on the fluorescence intensity of the TektdT signal. In AS mice, the brighttdT cell number increased and presented differential expression of endothelial markers compared to WT. RNA-seq analysis revealed differences in the immune and metabolic signaling pathways. In AS mice, dimtdT and brighttdT cells had different expression profiles of matrix-associated genes (Svep1, Itgß6), metabolic activity (Apom, Pgc1α) and immune modulation (Apelin, Icam1) compared to WT mice. We confirmed a new pro-inflammatory role of Apelin in AS mice and in cultured human GEC. Gene modulations were identified comparable to the biopsies from patients with AS and focal segmental glomerulosclerosis, possibly indicating that the same mechanisms apply to humans. We report the presence of two GEC subpopulations that differ between AS and healthy mice or humans. This finding paves the way to a better understanding of the pathogenic role of GEC in AS progression and could lead to novel therapeutic targets.

Circulating B Cells With Memory and Antibody-Secreting Phenotypes Are Detectable in Pediatric Kidney Transplant Recipients Before the Development of Antibody-Mediated Rejection.

Development of anti-human leukocyte antigen donor-specific antibodies (DSAs) is associated with antibody-mediated rejection (AMR) and reduced allograft survival in kidney transplant recipients. Whether changes in circulating lymphocytes anticipate DSA or AMR development is unclear. METHODS: We used time-of-flight mass cytometry to analyze prospectively collected peripheral blood mononuclear cells (PBMC) from pediatric kidney transplant recipients who developed DSA (DSA-positive recipients [DSAPOS], n = 10). PBMC were obtained at 2 months posttransplant, 3 months before DSA development, and at DSA detection. PBMC collected at the same time points posttransplant from recipients who did not develop DSA (DSA-negative recipients [DSANEG], n = 11) were used as controls. RESULTS: DSAPOS and DSANEG recipients had similar baseline characteristics and comparable frequencies of total B and T cells. Within DSAPOS recipients, there was no difference in DSA levels (mean fluorescence intensity [MFI]: 13 687 ± 4159 vs 11 375 ± 1894 in DSAPOSAMR-positive recipients (AMRPOS) vs DSAPOSAMR-negative recipients (AMRNEG), respectively; P = 0.630), C1q binding (5 DSAPOSAMRPOS [100%] vs 4 DSAPOSAMRNEG [80%]; P = 1.000), or C3d binding (3 DSAPOSAMRPOS [60%] vs 1 DSAPOSAMRNEG [20%]; P = 0.520) between patients who developed AMR and those who did not. However, DSAPOS patients who developed AMR (n = 5; 18.0 ± 3.6 mo post-DSA detection) had increased B cells with antibody-secreting (IgD-CD27+CD38+; P = 0.002) and memory (IgD-CD27+CD38-; P = 0.003) phenotypes compared with DSANEG and DSAPOSAMRNEG recipients at DSA detection. CONCLUSIONS: Despite the small sample size, our comprehensive phenotypic analyses show that circulating B cells with memory and antibody-secreting phenotypes are present at DSA onset, >1 year before biopsy-proven AMR in pediatric kidney transplant recipients.

T-cell exhaustion correlates with improved outcomes in kidney transplant recipients.

Continuous antigen stimulation during chronic infection or malignancy can promote functional T cell silencing, a phenomenon called T cell exhaustion. The prevalence and impact of T cell exhaustion following organ transplantation, another immune stimulus with persistently high antigen load, are unknown. Here, we characterized serially collected peripheral blood mononuclear cells from 26 kidney transplant recipients using time-of-flight mass cytometry (CyTOF) to define distinct subsets of circulating exhausted T cells and their relationship to induction therapy and allograft function. We observed an increase in specific subsets of CD4+ and CD8+ exhausted T cells from pre-transplant to 6-months post-transplant, with greater increases in participants given anti-thymocyte globulin induction than in participants who received no induction or non-depleting induction. The percentages of exhausted T cells at 6 months correlated inversely with adenosine triphosphate (ATP) production (a surrogate of T cell function) and with allograft interstitial fibrosis. Guided by the CyTOF data, we delineated a PD-1+CD57- phenotype for CD4+ and CD8+ exhausted T cells, and confirmed that these cells have limited capacity for cytokine secretion and ATP production. In an independent cohort of 50 kidney transplant recipients, we confirmed the predicted increase of PD-1+CD57- exhausted T cells after lymphocyte-depleting induction therapy and its direct correlation with better allograft function. Our findings suggest that monitoring T cell exhaustion can be useful for post-transplant risk assessment and support the need to develop and test strategies aimed at augmenting T cell exhaustion following kidney transplantation.

Concise Reviews: Stem Cells and Kidney Regeneration: An Update.

Significant progress has been made to advance stem cell products as potential therapies for kidney diseases: various kinds of stem cells can restore renal function in preclinical models of acute and chronic kidney injury. Nonetheless this literature contains contradictory results, and for this reason, we focus this review on reasons for apparent discrepancies in the literature, because they contribute to difficulty in translating renal regenerative therapies. Differences in methodologies used to derive and culture stem cells, even those from the same source, in addition to the lack of standardized renal disease animal models (both acute and chronic), are important considerations underlying contradictory results in the literature. We propose that harmonized rigorous protocols for characterization, handling, and delivery of stem cells in vivo could significantly advance the field, and present details of some suggested approaches to foster translation in the field of renal regeneration. Our goal is to encourage coordination of methodologies (standardization) and long-lasting collaborations to improve protocols and models to lead to reproducible, interpretable, high-quality preclinical data. This approach will certainly increase our chance to 1 day offer stem cell therapeutic options for patients with all-too-common renal diseases. Stem Cells Translational Medicine 2019;8:82-92.

Impact of preformed T-cell alloreactivity by means of donor-specific and panel of reactive T cells (PRT) ELISPOT in kidney transplantation.

Donor-specific (d-sp) interferon gamma enzyme-linked immunosorbent spot (d-sp ELISPOT) and Panel of reactive T-cell (PRT) ELISPOT assays have been developed to detect alloreactive memory T (Tmem) cells in order to estimate the risk of acute rejection after kidney transplantation. Adding IL15 to the PRT assay (PRT+IL15) may uncover the presence of pathogenic alloreactive CD28-Tmem. Face-to-face comparisons of these assays have not been done yet. We performed pre-transplant d-sp ELISPOT and PRT assays (±IL15, against six B-cell lines) in 168 consecutive kidney transplant recipients and evaluated the multivariable-adjusted associations with biopsy-proven acute rejection (BPAR), de novo donor-specific antibodies (DSA), and eGFR decline over a 48-month follow-up period. D-sp ELISPOT was positive in 81 (48%) subjects, while 71 (42%) and 81 (48%) subjects displayed positive PRT and PRT+IL15, respectively. Their median [interquartile range] numerical test result was 23 [6-65], 18 [8-37], and 26 [10-45] spots/3x105 PBMCs, respectively. The number of PRT spots were weakly correlated with those of d-sp ELISPOT, but highly correlated with PRT+IL15 (rho = 0.96, P<0.001). d-sp ELISPOT, but not PRT (±IL15) was independently associated with BPAR (adjusted Odds Ratio of BPAR associated with d-sp ELISPOT positivity: 4.20 [95%CI: 1.06 to 21.73; P = 0.041]). Unlike d-sp ELISPOT, median PRT and PRT+IL15 were independently associated with higher Δ3-48month eGFR decline post-transplantation (for both assays, about -3mL/min/1.73m2 per one standard deviation unit increase in the spot number). Pre-transplant T-cell immune-monitoring using d-sp ELISPOT and PRT assays identifies kidney transplant candidates at high risk of BPAR and worse kidney allograft progression.

Amniotic fluid cells: current progress and emerging challenges in renal regeneration.

Amniotic fluid (AF) contains a heterogeneous population of cells that have been identified to possess pluripotent and progenitor-like characteristics. These cells have been applied in various regenerative medicine applications ranging from in vitro cell differentiation to tissue engineering to cellular therapies for different organs including the heart, the liver, the lung, and the kidneys. In this review, we examine the different methodologies used for the derivation of amniotic fluid stem cells and renal progenitors, and their application in renal repair and regeneration. Moreover, we discuss the recent achievements and newly emerging challenges in our understanding of their biology, their immunoregulatory characteristics, and their paracrine-mediated therapeutic potential for the treatment of acute and chronic kidney diseases.

Thyroid Hormone Receptor Alpha is Essential to Maintain the Satellite Cell Niche During Skeletal Muscle Injury and Sarcopenia of Aging.

BACKGROUND: Myopathic changes are commonly described in hypothyroid and hyperthyroid patients, including muscular atrophy and weakness. Satellite cells (SCs) play a major role in skeletal muscle maintenance and regeneration after injury. A mouse model of resistance to thyroid hormone-TRα1PV demonstrated impaired skeletal muscle regeneration after injury with significant reduction of SCs, suggesting that exhaustion of the SC pool contributes to the impaired regeneration. To test this hypothesis, SC activation and proliferation were analyzed in vivo in response to skeletal muscle injury and during aging. METHODS: SCs of TRα1PV male mice were analyzed four days after cardiotoxin-induced muscle injury, and they were compared to wild-type (WT) male animals. TRα-knockdown C2C12 myoblasts were injected into injured skeletal muscle, and four days after transplantation, the in vivo behavior was compared to control C2C12 myoblasts. Skeletal muscle regeneration was compared in younger and older TRα1PV and WT animals. RESULTS: The total number of SCs in skeletal muscle of TRα1PV mice was significantly lower than control, both before and shortly after muscle injury, with significant impairment of SC activation, consistent with SC pool exhaustion. TRα-knockdown myoblasts showed impaired in vivo proliferation and migration. TRα1PV mice had skeletal muscle loss and significant impairment in skeletal muscle regeneration with aging. This translated to a significant reduction of the SC pool with aging compared to WT mice. CONCLUSION: TRα plays an important role in the maintenance of the SC pool. Impaired skeletal muscle regeneration in TRα1PV mice is associated with insufficient SC activation and proliferation, as well as the progressive loss of the SC pool with aging. Regulation of the SC pool and SC proliferation provides a therapeutic target to enhance skeletal muscle regeneration and possibly slow age-associated sarcopenia.

A step towards clinical application of acellular matrix: A clue from macrophage polarization.

The outcome of tissue engineered organ transplants depends on the capacity of the biomaterial to promote a pro-healing response once implanted in vivo. Multiple studies, including ours, have demonstrated the possibility of using the extracellular matrix (ECM) of animal organs as platform for tissue engineering and more recently, discarded human organs have also been proposed as scaffold source. In contrast to artificial biomaterials, natural ECM has the advantage of undergoing continuous remodeling which allows adaptation to diverse conditions. It is known that natural matrices present diverse immune properties when compared to artificial biomaterials. However, how these properties compare between diseased and healthy ECM and artificial scaffolds has not yet been defined. To answer this question, we used decellularized renal ECM derived from WT mice and from mice affected by Alport Syndrome at different time-points of disease progression as a model of renal failure with extensive fibrosis. We characterized the morphology and composition of these ECMs and compared their in vitro effects on macrophage activation with that of synthetic scaffolds commonly used in the clinic (collagen type I and poly-L-(lactic) acid, PLLA). We showed that ECM derived from Alport kidneys differed in fibrous protein deposition and cytokine content when compared to ECM derived from WT kidneys. Yet, both WT and Alport renal ECM induced macrophage differentiation mainly towards a reparative (M2) phenotype, while artificial biomaterials towards an inflammatory (M1) phenotype. Anti-inflammatory properties of natural ECMs were lost when homogenized, hence three-dimensional structure of ECM seems crucial for generating an anti-inflammatory response. Together, these data support the notion that natural ECM, even if derived from diseased kidneys promote a M2 protolerogenic macrophage polarization, thus providing novel insights on the applicability of ECM obtained from discarded organs as ideal scaffold for tissue engineering.

Thyroid Hormone Receptor α Plays an Essential Role in Male Skeletal Muscle Myoblast Proliferation, Differentiation, and Response to Injury.

Thyroid hormone plays an essential role in myogenesis, the process required for skeletal muscle development and repair, although the mechanisms have not been established. Skeletal muscle develops from the fusion of precursor myoblasts into myofibers. We have used the C2C12 skeletal muscle myoblast cell line, primary myoblasts, and mouse models of resistance to thyroid hormone (RTH) α and ß, to determine the role of thyroid hormone in the regulation of myoblast differentiation. T3, which activates thyroid hormone receptor (TR) α and ß, increased myoblast differentiation whereas GC1, a selective TRß agonist, was minimally effective. Genetic approaches confirmed that TRα plays an important role in normal myoblast proliferation and differentiation and acts through the Wnt/ß-catenin signaling pathway. Myoblasts with TRα knockdown, or derived from RTH-TRα PV (a frame-shift mutation) mice, displayed reduced proliferation and myogenic differentiation. Moreover, skeletal muscle from the TRα1PV mutant mouse had impaired in vivo regeneration after injury. RTH-TRß PV mutant mouse model skeletal muscle and derived primary myoblasts did not have altered proliferation, myogenic differentiation, or response to injury when compared with control. In conclusion, TRα plays an essential role in myoblast homeostasis and provides a potential therapeutic target to enhance skeletal muscle regeneration.

Tissue-Engineering Approaches to Restore Kidney Function.

Kidney transplantation for the treatment of chronic kidney disease has established outcome and quality of life. However, its implementation is severely limited by a chronic shortage of donor organs; consequently, most candidates remain on dialysis and on the waiting list while accruing further morbidity and mortality. Furthermore, those patients that do receive kidney transplants are committed to a life-long regimen of immunosuppressive drugs that also carry significant adverse risk profiles. The disciplines of tissue engineering and regenerative medicine have the potential to produce alternative therapies which circumvent the obstacles posed by organ shortage and immunorejection. This review paper describes some of the most promising tissue-engineering solutions currently under investigation for the treatment of acute and chronic kidney diseases. The various stem cell therapies, whole embryo transplantation, and bioengineering with ECM scaffolds are outlined and summarized.

A Periodic Diet that Mimics Fasting Promotes Multi-System Regeneration, Enhanced Cognitive Performance, and Healthspan.

Prolonged fasting (PF) promotes stress resistance, but its effects on longevity are poorly understood. We show that alternating PF and nutrient-rich medium extended yeast lifespan independently of established pro-longevity genes. In mice, 4 days of a diet that mimics fasting (FMD), developed to minimize the burden of PF, decreased the size of multiple organs/systems, an effect followed upon re-feeding by an elevated number of progenitor and stem cells and regeneration. Bi-monthly FMD cycles started at middle age extended longevity, lowered visceral fat, reduced cancer incidence and skin lesions, rejuvenated the immune system, and retarded bone mineral density loss. In old mice, FMD cycles promoted hippocampal neurogenesis, lowered IGF-1 levels and PKA activity, elevated NeuroD1, and improved cognitive performance. In a pilot clinical trial, three FMD cycles decreased risk factors/biomarkers for aging, diabetes, cardiovascular disease, and cancer without major adverse effects, providing support for the use of FMDs to promote healthspan.

Renal Extracellular Matrix Scaffolds From Discarded Kidneys Maintain Glomerular Morphometry and Vascular Resilience and Retains Critical Growth Factors.

BACKGROUND: Extracellular matrix (ECM) scaffolds, obtained through detergent-based decellularization of native kidneys, represent the most promising platform for investigations aiming at manufacturing kidneys for transplant purposes. We previously showed that decellularization of the human kidney yields renal ECM scaffolds (hrECMs) that maintain their basic molecular components, are cytocompatible, stimulate angiogenesis, and show an intact innate vasculature. However, evidence that the decellularization preserves glomerular morphometric characteristics, physiological parameters (pressures and resistances of the vasculature bed), and biological properties of the renal ECM, including retention of important growth factors (GFs), is still missing. METHODS: To address these issues, we studied the morphometry and resilience of hrECMs' native vasculature with resin casting at electronic microscopy and pulse-wave measurements, respectively. Moreover, we determined the fate of 40 critical GFs post decellularization with a glass chip-based multiplex enzyme-linked immunosorbent assay array and in vitro immunofluorescence. RESULTS: Our method preserves the 3-dimensional conformation of the native glomerulus. Resin casting and pulse-wave measurements, showed that hrECMs preserves the microvascular morphology and morphometry, and physiological function. Moreover, GFs including vascular endothelial growth factor and its receptors are retained within the matrices. CONCLUSIONS: Our results indicate that discarded human kidneys are a suitable source of renal scaffolds because they maintain a well-preserved structure and function of the vasculature, as well as GFs that are fundamental to achieve a satisfying recellularization of the scaffold in vivo due to their angiogenic properties.